Cells were sorted (BD DiVa) based on tdTomato, CD24, EpCam, and CD45 as indicated in the figure legend then RNA was prepped from at least (Mock: 78,000, 2 DPI: 4,100, 14 DPI SC: 44,000, 14 DPI: 100,000) cells using RNeasy Plus Mini kit (74104). Samples were then prepped using NEBNext Poly(A) messenger RNA (mRNA) Magnetic Isolation Module (NEB #E74905). NEBNext Ultra II RNA Library Prep Kit (E7770S) was used to prep libraries with NEBNext Multiplex Oligos for Illumina (E7335S, E7500S) for adaptor ligation and enrichment of libraries. Samples were analyzed on two lanes of Illumina HiSeq 2500 in rapid run mode. The raw and normalized data is accessible at NCBI GEO under the accession number GSE116032. The reads were mapped to the mouse transcriptome (mm10) and the B/Mal/04 genome using Bowtie, and samples were run on Basespace (Illumina). Finally reads were imported into DeSeq2 for analysis65 (link).
Nebnext ultra 2 rna library prep kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Ultra II RNA Library Prep Kit is a reagent kit designed for the preparation of RNA libraries for next-generation sequencing. The kit provides a streamlined workflow for generating high-quality libraries from total RNA samples.
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Lab products found in correlation
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (14 mentions)
Novaseq 6000, by Illumina (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Nextseq 500, by Illumina (9 mentions)
Hiseq 2500, by Illumina (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Nextseq 500 platform, by Illumina (6 mentions)
Bioanalyzer, by Agilent Technologies (5 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Fragment analyzer, by Agilent Technologies (5 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (5 mentions)
Rneasy kit, by Qiagen (4 mentions)
Nebnext rrna depletion kit, by New England Biolabs (4 mentions)
Hiseq 4000, by Illumina (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (4 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nextseq 500 sequencer, by Illumina (3 mentions)
101 protocols using nebnext ultra 2 rna library prep kit
1
Single-cell RNA-seq of influenza infection
2
Transcriptomic profiling of limb development
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Pairs of mouse forelimb buds at E9.75 (28–30 somites), E10.5 (34–36 somites), and E11.5 (43–45 somites) and chicken wing buds at HH20, HH22, and HH24 were dissected in ice-cold PBS and transferred to RNAlater (Sigma), stored at 4 °C overnight and then transferred to −20 °C. Tissue homogenization in RLT buffer (Qiagen) was done by sheering the sample with a 25 G needle 20 times. Total RNA was extracted using the RNeasy micro kit (Qiagen) including an on-column DNAse digestion (Qiagen) step to minimize the genomic DNA contamination. RNA integrity was checked on an Agilent Fragment Analyser and the concentration was determined using the QubitTM RNA HS Assay kit (Ref Q32855). About 500 ng of total RNA was used to prepare RNA-seq libraries with NEB nondirectional NEBNext® Ultra™ II RNA Library Prep kit by applying the polyA+ mRNA workflow and PCR amplification for nine cycles. Barcoded RNA-seq libraries were pooled at equimolar concentrations and sequenced as single-end using Illumina NextSeq 500.
3
RNA-seq Library Preparation and Analysis
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The extracted mRNA was used to construct RNA-seq libraries using rRNA-depleted RNAs with the NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s protocol. To ensure that the sample quality requirements were met before testing, library quantification was performed using a Qubit 2.0 instrument, and library insert size was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were then pooled and sequenced on the HiSeqTM 2500 system (Illumina, San Diego, CA, USA). Gene expression levels were estimated according to the expected number of fragments per kilobase of transcript per million mapped fragments by HTSeq v0.9.1 (Anders et al., 2015 (link)). Pairwise comparisons were performed, and clustering was implemented for all expressed genes using the Trinity v2.8.4 package (Grabherr et al., 2011 (link)).
4
ChIRP-seq analysis of LLNLR-299G3.1 lncRNA
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ChIRP-seq (Chromatin isolation by RNA purification sequencing) experiments were performed as previously described with minor modifications [20 (link)]. In brief, seveal DNA probes against the LLNLR-299G3.1 full-length sequence were ordered from Dianxi Biotech (Shanghai, China) and were biotinylated at the 3′ end. TE1 cells were cross-linked, lysed sonicated, incubated with probe sets, and LLNLR-299G3.1-bound chromatin was retrieved. The ChIRPed RNA was purified with proteinase K buffer and then subjected for library construction using the NEBNext Ultra II RNA Library Prep kit (New England Biolabs, MA, USA). The purified DNA fragments were sequenced on an Illumina Novaseq PE150 system at Dianxi Biotech (Shanghai, China). Raw reads were mapped to human reference genome assembly (GRCh38) using the STAR (Spliced Transcripts Alignment to a Reference) version 2 software. Peaks were called using the MACS package. Motif enrichment analysis was performed using the online tool Homer (http://homer.ucsd.edu/homer/ngs/peakMotifs.html ). The sequences of probes for LLNLR-299G3.1 are listed in Suppl. Table S1.
5
Illumina RNA Sequencing Library Prep
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Total RNA was isolated using Trizol (Life Technologies) according to the manufacturer’s instructions. The NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs) for Illumina workflow was used to generate the sequencing libraries. The specific library construction process is described online at www.neb.com/products/e7770-nebnext-ultra-ii-rna-library-prep-kit-for-illumina .
6
RNA-seq of SUDHL-4 and LoVo cells
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For RNA-seq sample preparation, 5–10×10^6 SUDHL-4 or LoVo cells (untreated, UNC7263 or UNC7040 treated in triplicates) were trypsinized and collected by centrifugation for 5 min at 500 g. Subsequently, cell pellets were washed 1× with PBS and collected in 1X DNA/RNA protection reagent (Monarch Total RNA Miniprep Kit, NEB). Cells were lysed and total RNA was isolated following the mammalian cell protocol inlcuding on-column DNase I treatment. Total RNA samples were submitted to Novogene Co. for quality control and library preparation, applying poly-A enrichment and using the NEBNext Ultra II RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB). Final libraries were sequenced as 150 bp paired-end reads on the Illumina HiSeq platform.
7
RNA Isolation and Library Preparation
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Total RNA was isolated using an RNeasy mini kit (Qiagen, Hilden, Germany) from PBMCs following 100 ng/ml LPS and 25 µM PNLA or vehicle treatment. RNA was purified using an RNeasy kit on-column with DNase I digestion (Qiagen) as described in Supplementary Data S3 (available at Rheumatology online). The cell lysates were stored at −80°C and then passed through a series of spin columns to first bind genomic DNA, then RNA and finally to elute high-quality RNA. A quality control check for RNA was assessed as described in Supplementary Data S3 (available at Rheumatology online).
Library preparation including ribosomal RNA depletion using NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) for Illumina, as described inSupplementary Data S3 (available at Rheumatology online).
Library preparation including ribosomal RNA depletion using NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) for Illumina, as described in
8
RNA-seq Analysis of Phage-infected Bacteria
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Total RNA was extracted from the indicated strains and conditions 2 h post phage infection using TRIzol. The integrity of the total cellular RNA was evaluated using RNA tape of Agilent TapeStation 2200 before library preparation. All RNA samples were of high integrity with a RIN score of 7.0 or more. rRNA was first depleted from 500 ng of each sample using MICROBExpress Kit (AM1905, Fisher) following the manufacture’s instruction. The rRNA-depleted total RNA was subjected to library preparation using NEBNext® Ultra™ II RNA Library Prep Kit (E7700, New England Biolabs) and barcoded with NEBNext Multiplex Oligos for Illumina (E7730, New England Biolabs) following the manufacturer’s instructions. The libraries were pooled with equal amounts of moles, further sequenced using MiSeq Reagent V3 (MS-102-3003, Illumina) for pair-ended, 600-bp reads, and demultiplexed using the build-in bcl2fastq code in Illumina sequence analysis pipeline. Raw sequencing reads have been deposited as part of BioProject PRJNA806967 in the NCBI SRA database.
9
RNA-Seq Library Preparation and Analysis
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Total RNA was extracted from cells using Omega Total RNA Kit I accordingly to the manufacturer’s recommended procedures. Poly(A) RNA enrichment was conducted using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), and the sequencing library was constructed by using the NEBNext Ultra II RNA Library Prep Kit (NEB) following the manufacturer’s instructions. The resulting library was subjected to sequencing analysis on an MGI 2000 platform. The sequencing reads were aligned to GRCh38 assembly using STAR (v.2.7.0) with default parameters (33 (link)). Transcript quantification was conducted using featureCounts (v.2.0.3) (34 (link)). Differential gene expression analysis was performed with DESeq2 (v.1.36.0) (35 (link)).
10
Bulk RNA-seq Data Processing Protocol
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Total RNA was extracted using a PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then mRNAs were used for library construction by using the NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s recommendations. Libraries were quantified using the 2200 TapeStation (Agilent, Santa Clara, CA, USA) and sequenced using the Illumina HiSeq 3000 or Novaseq S2.
All the sequenced raw reads were filtered and trimmed with Trim Galore (v0.4.1) before subsequent analysis. For bulk RNA-seq data processing, the clean reads were mapped to the human genome (GRC38) using Hisat2 [28 (link)]. Then, the number of reads uniquely mapped to each gene was determined using FeatureCounts from subread2 [29 (link)]. The differentially expressed genes (DEGs) were identified by DESeq2 [30 (link)] with p values less than 0.01. Principal component analysis (PCA) was performed using all the genes after removing the batch effect by DESeq2.
All the sequenced raw reads were filtered and trimmed with Trim Galore (v0.4.1) before subsequent analysis. For bulk RNA-seq data processing, the clean reads were mapped to the human genome (GRC38) using Hisat2 [28 (link)]. Then, the number of reads uniquely mapped to each gene was determined using FeatureCounts from subread2 [29 (link)]. The differentially expressed genes (DEGs) were identified by DESeq2 [30 (link)] with p values less than 0.01. Principal component analysis (PCA) was performed using all the genes after removing the batch effect by DESeq2.
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